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The Tonb System in Aeromonas Hydrophila NJ-35 Is Essential for Maca2b2 Efflux Pump-Mediated Macrolide Resistance

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The Tonb System in Aeromonas Hydrophila NJ-35 Is Essential for Maca2b2 Efflux Pump-Mediated Macrolide Resistance Dong et al. Vet Res (2021) 52:63 https://doi.org/10.1186/s13567-021-00934-w RESEARCH ARTICLE Open Access The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efux pump-mediated macrolide resistance Yuhao Dong1, Qing Li2, Jinzhu Geng1, Qing Cao1, Dan Zhao1, Mingguo Jiang3, Shougang Li1, Chengping Lu1 and Yongjie Liu1* Abstract The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a signifcantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no efect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the dele- tion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicat- ing that the macrolide resistance aforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any infuence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system signifcantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efux. Keywords: Aeromonas hydrophila, TonB system, Macrolides, MacA2B2 efux pump, Sensitivity Introduction such as iron and vitamins that are present in the extracel- Gram-negative bacteria comprise most of the bacterial lular environment at very low concentrations [3]. Te sol- world. Diferent from Gram-positive bacteria, which utes that have a molecular mass less than 600 Da can pass only have a cytoplasmic membrane (CM), the cell enve- through OM porins by the concentration-gradient-driven lope of Gram-negative bacteria contains two membranes, passive difusion [4]. However, the uptake of nutrients, namely the CM and the outer membrane (OM) [1]. Te especially those existing at extremely low concentrations, OM protects Gram-negative bacteria from environmen- must occur by OM active transport. Due to the cell enve- tal hazards such as antibiotics and detergents [2]. Also, lope architecture, there is no electrochemical gradient Gram-negative bacteria require rare essential nutrients powering the active transport across the OM and no ATP in the periplasmic space, which means that OM trans- *Correspondence: [email protected] porters need to extract energy from the CM. Te energy 1 Joint International Research Laboratory of Animal Health and Food transfer is commonly carried out by trans-periplasmic Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China proteins, for example, the CM-anchored TonB complex Full list of author information is available at the end of the article [5]. TonB system, consisting of TonB, ExbB and ExbD © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Dong et al. Vet Res (2021) 52:63 Page 2 of 10 proteins, couples the proton motive force (PMF) of the A. hydrophila and E. coli were routinely grown in CM to energize active transport across the OM by the Luria–Bertani (LB) medium at 28 °C and 37 °C, respec- TonB-dependent transporters (TBDTs) [6]. TonB pro- tively. When necessary, the medium was supplemented tein, the main component of this system, is CM-anchored with the following antibiotics: chloramphenicol (Cm), by a transmembrane helix, and stabilized by ExbB and 34 mg/L for E. coli; ampicillin (Amp), 100 mg/L for A. ExbD [7]. TonB system was thought to be only involved hydrophila. All reagents used in this study were sup- in nutrient import. In Escherichia coli, TonB system pro- plied by Sigma (St. Louis, MO, USA) unless otherwise vides energy to the OM protein BtuB for vitamin B 12 indicated. transport [3]. In Vibrio anguillarum, TonB1 system is responsible for heme and ferrichrome transport, while Minimum inhibitory concentration (MIC) assay TonB2 system is essential for the transport of endoge- A. hydrophila NJ-35 and its derivative tonB123 mutant nous and exogenous siderophores [8]. However, it is now grown to logarithmic phase were assayed for macrolide becoming clear that the uptake of nutrient is only one of sensitivity. MICs were determined by broth microdilu- the many potential functions of the TonB system. Indeed, tion, following the Clinical and Laboratory Standards in Myxococcus xanthus, TonB system is required for the Institute (CLSI) guidelines [14]. Briefy, cultured cells in 5 secretion of the protease PopC, which suggests that TonB the log phase were diluted to 2 × ­10 cells/mL in fresh system not just participates in import processes, but also Mueller–Hinton broth (MHB). Te inoculum (100 μL) is involved in the secretion of intracellular proteins [9]. was added to each well of 96-well plates. Antibiotic was Aeromonas hydrophila is one of the most important then added to the frst wells and twofold dilutions were fsh pathogens causing haemorrhagic septicaemia. Also, performed. Plates were incubated at 28 °C for 18 h. Te this bacterium is associated with a variety of human MIC value was measured three times and the average of illnesses, such as sepsis, wound infections, and food- the measured values was determined as the MIC value borne gastroenteritis [10]. To date, antibiotics are still for the strain and the antibiotic. Macrolide antibiotics, the most efective way to fght this bacterial infection. including roxithromycin (ROX), erythromycin (ERY), However, the widespread use of antibiotics has led to the tilmicosin (TIL), tylosin (TYL), acetylspiramycin (ACE), emergence of antimicrobial resistance in A. hydrophila azithromycin (AZI), dirithromycin (DIR) and medemycin and bioaccumulation in host tissues, which seriously (MED) were purchased from Solarbio (Beijing, China). threatened human and public health. Some strains of A. To determine whether iron was involved in the efect hydrophila have been found to be resistant to macrolides, on the bacterial sensitivity to macrolides, MIC of each tetracyclines, sulfonamides, and quinolones [11, 12]. In macrolide was examined in MHB supplemented with response to this phenomenon, understanding of resist- 36 μM FeCl3 or 150 μM 2, 2-dipyridyl (DIP). ance mechanisms has become an urgent necessity for development of an efective therapeutic strategy against Cell membrane integrity this pathogen. To determine whether deletion of tonB123 resulted in Recently, three tonB genes in A. hydrophila NJ-35, damage to the permeability barrier of the bacterial cell namely tonB1, tonB2 and tonB3, were described to be membrane, cell membrane integrity was examined by involved in antibiotic resistance. A triple-deletion mutant determining the release of cytoplasmic constituents into of tonB123 showed a signifcantly increased sensitivity to the supernatant, such as nucleic acids and proteins [15]. the macrolide antibiotics erythromycin and roxithromy- Te bacteria were incubated at 28 °C for 6 h, and then cin, but had no efect on other classes of antibiotics [13]. immediately centrifuged (9000 g) for 5 min at 4 °C. Te In this study, we further demonstrated that the increased supernatant was flter-sterilized using 0.22-µm (pore- susceptibility of ΔtonB123 mutant to macrolides was due size) membrane. Te amount of nucleic acids released to the decrease in drug efux, and furthermore, associ- from the cytoplasm was determined by measuring the ated with the MacA2B2-mediated pump. optical density at 260 nm. Te concentration of proteins in the supernatant was determined using Protein Brad- ford Assay kit (Termo Fisher Scientifc, Waltham, USA). Materials and methods Bacterial strains and growth conditions Morphological observation Te bacterial strains and plasmids used in this study are Bacterial cell morphology was evaluated using scan- listed in Additional fle 1. A. hydrophila NJ-35 (accession ning electron microscope (SEM) and transmission elec- number CP006870), which belongs to the ST251 clonal tron microscope (TEM) [16]. For SEM examination, the group, was isolated from dead cultured cyprinoid fsh in dehydrated samples were treated thrice with 100% tert- the Jiangsu province of China in 2010. butanol and dried with a freeze dryer for 2 h. Te samples Dong et al. Vet Res (2021) 52:63 Page 3 of 10 were placed on stubs and coated with gold flm by sputter chromosomal DNA of A. hydrophila NJ-35, and then coating and viewed using a FEI Quanta FEG250 scanning ligated in-frame using fusion PCR.
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